leupeptins and Breast-Neoplasms

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for i

Topics: Animals; Breast; Breast Neoplasms; Centrifugation, Density Gradient; Cytosol; Drug Resistance; Endometrium; Endopeptidases; Female; Humans; Kidney; Leukemia; Leupeptins; Liver; Lysine; Macromolecular Substances; Male; Molybdenum; Osmolar Concentration; Protein Conformation; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Glucocorticoid; Receptors, Progesterone; Substrate Specificity; Ultracentrifugation; Uterine Neoplasms

Breast cancer is the most commonly diagnosed malignant tumor among females. Estrogen receptor α (ERα) is initially expressed in 70% of breast cancers and is a well-known target of endocrine therapy for ERα-positive breast cancer. In the present study, we identified MINDY1, a member belongs to the motif interacting with Ubcontaining novel DUB family (MINDY), as a potential deubiquitylase of ERα in breast cancer. There was a positive correlation between ERα and MINDY1 protein levels in human breast cancer tissues. We found that high expression of MINDY1 was associated with poor prognosis. MINDY1 interacted with ERα, thereby mediating the deubiquitination of ERα and increased its stability in a deubiquitylation activity-dependent manner. MINDY1 depletion significantly decreased the ERα protein level and ERα signaling activity in breast cancer cells. Specifically, MINDY1 associated with the N-terminal of ERα via its catalytic domain, thus inhibiting K48-specific poly-ubiquitination process on ERα protein. In addition, MINDY1 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells. Finally, overexpression of ERα could rescue the MINDY1 depletion-induced growth inhibition both in vitro and in vivo, suggesting that MINDY1 promotes breast carcinogenesis through increasing ERα stability. Overall, our study proposed a novel post-translational mechanism of ERα in supporting breast cancer progression. Targeting the MINDY1 may prove to be a promising strategy for patients with ERα-positive breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Deubiquitinating Enzymes; Estrogen Receptor alpha; Female; HEK293 Cells; Humans; Leupeptins; Mice, Inbred BALB C; Mice, Nude; Prognosis; Proteasome Inhibitors; Protein Binding; Protein Stability; RNA, Small Interfering; Signal Transduction; Ubiquitination

The histone H3K9 methyltransferase SETDB2 is involved in cell cycle dysregulation in acute leukemia and has oncogenic roles in gastric cancer. In our study, we found that SETDB2 plays essential roles in breast cancer stem cell maintenance. Depleted SETDB2 significantly decreased the breast cancer stem cell population and mammosphere formation

Topics: Breast Neoplasms; Cell Line, Tumor; Cycloheximide; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Histone-Lysine N-Methyltransferase; Humans; Leupeptins; Neoplastic Stem Cells; Transcription Factors; Tumor Suppressor Proteins

Functional imaging has become an important tool in oncology because it not only provides information about the size and localization of the tumour, but also about the pathophysiological features of the tumoural cells. One of the characteristic features of some tumour types is that their fast growth leads to deficient intratumoral vascularization, which results in low oxygen availability. To overcome this lack of oxygen, tumoural cells activate the neoangiogenic program by upregulating the transcription factor HIF-1α. Herein we report a non-invasive in vitro detection method of hypoxia using designed fluorescent peptide probes based on the oxygen-dependent degradation domain of HIF-1α. The fluorescent probe retains the oxygen-sensing capability of HIF-1α, so that it is stabilized under hypoxia and readily degraded by the proteasome under normoxia, thus providing direct information of the cellular oxygen availability.

Topics: Animals; Biosensing Techniques; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cobalt; Female; Fluorescent Dyes; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Mice; Mice, Inbred BALB C; Mice, Nude; Mimosine; Oxygen; Peptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Domains; Proteolysis; Spectrometry, Fluorescence

BACKGROUND Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), a peptide aldehyde proteasome inhibitor, can inhibit tumor progression by inactivating nuclear factor (NF)-κB signaling. Paclitaxel (PTX) is part of a routine regimen for the treatment of breast cancer. However, activation of the NF-κB pathway after treatment with PTX confers insensitivity to this drug. This study investigated the potential effect of MG132 as a co-treatment with PTX against breast cancer, and clarifies the underlying molecular mechanisms. MATERIAL AND METHODS Breast cancer cells were treated with PTX, MG132, or PTX plus MG132, and the therapeutic effects were evaluated phenotypically. A mouse model of breast cancer was used to determine the combined effect of PTX plus MG132 in vivo. RESULTS Treatment with PTX plus MG132 suppressed aggressive phenotypes of breast cancer cells more effectively than PTX alone. Consistently, MG132 also enhanced the suppressive effect of PTX on tumor growth in C57BL/6 mice. Significantly, activation of the NF-κB pathway by PTX was attenuated by MG132. CONCLUSIONS Based on our findings, we suggest the application of MG132 in clinical practice in combination with PTX for the treatment of breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Movement; Cell Proliferation; Cell Survival; Female; Human Umbilical Vein Endothelial Cells; Humans; Leupeptins; MCF-7 Cells; Mice, Inbred C57BL; Neoplasm Invasiveness; NF-kappa B; Paclitaxel; Phenotype; Proteasome Inhibitors; Signal Transduction

Macrophage reprogramming toward a tumor-attacking phenotype is a promising treatment strategy, yet such strategies are scarce and it is not clear how to combine them with cytotoxic therapies that are often used to treat solid tumors. Here, we evaluate whether a micelle-encapsulated proteasome inhibitor, that is, the peptide aldehyde drug MG132, which is cytotoxic to cancer cells, can reprogram macrophages to attack the tumor. Through in vitro studies, we demonstrated that the proteasome inhibition reduces nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling-a known promoter of tumor-supporting macrophages and chemoresistance-in both cancer cells and macrophages. In in vivo studies, we showed that, although free MG132 did not affect the macrophage phenotype in tumors even at its maximum tolerated dose, the micellar formulation of MG132 safely achieved simultaneous cancer cell killing and macrophage reprogramming, thereby enhancing the antitumor efficacy in a syngeneic, orthotopic breast cancer model.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Polarity; Cellular Reprogramming; Female; Leupeptins; Macrophages; Mice, Inbred BALB C; Micelles; NF-kappa B; Proteasome Inhibitors; Xenograft Model Antitumor Assays

Protein kinase C (PKC)-eta (PKCη) is a member of the novel category of PKC family. It is overexpressed in breast cancer and was shown to inhibit apoptosis and contribute to chemoresistance. Since the anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance, we investigated if PKCη regulates Mcl-1 level. Silencing of PKCη decreased Mcl-1 in several breast cancer cells, including MCF-7 and T47D cells. PKCη depletion had no effect on MCL1 mRNA but the decrease in Mcl-1 by PKCη knockdown was blocked by proteasomal inhibitors, such as MG132 and lactacystin. Moreover, knockdown of Mule (Mcl-1 ubiquitin ligase) prevented Mcl-1 downregulation caused by PKCη deficiency. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-β, a substrate for Akt, had little effect on Mcl-1 downregulation caused by PKCη silencing. However, knockdown of PKCη but not PKCα, -δ or -ε caused a significant decrease in ERK (extracellular signal-regulated kinase) phosphorylation. Knockdown of ERK1 but not ERK2 decreased Mcl-1 level, and the decrease in Mcl-1 caused by PKCη knockdown was restored by ERK1 overexpression. These results suggest that PKCη utilizes the ERK signaling pathway to protect against ubiquitin-mediated proteasomal degradation of Mcl-1.

Topics: Apoptosis; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Glycogen Synthase Kinase 3 beta; Humans; Leupeptins; MAP Kinase Signaling System; MCF-7 Cells; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myeloid Cell Leukemia Sequence 1 Protein; Protein Kinase C; Protein Kinase C-alpha

DNA damage activated by Adriamycin (ADR) promotes ubiquitin-proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Female; Humans; Leupeptins; MCF-7 Cells; Proteasome Inhibitors; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53; Ubiquitination

EZH2 (the Enhancer of Zeste Homolog 2), as a key epigenetic regulator and EMT inducer, participates in a variety of cancer metastasis. EZH2 stability is regulated by several types of post-translational modifications (PTMs).The long non-coding RNAs (lncRNA) have been implicated to have critical roles in multiple carcinogenesis through a wide range of mechanisms, including modulating the stability of proteins. To date, whether the stability of EZH2 protein is regulated by lncRNAs remains unexplored. Here we report the discovery of ANCR modulating the stability of EZH2, and hence in the invasion and metastasis of breast cancer cells. We determined that ANCR potentiated the CDK1-EZH2 interaction, which then increased the intensity of phosphorylation at Thr-345 and Thr-487 sites of EZH2, facilitating EZH2 ubiquitination and hence its degradation. Moreover, we also uncover ANCR is an important player in breast cancer progression and metastasis mainly through decreasing EZH2 stability. More specifically, we initially found that ANCR level was lower in breast cancer tissues and breast cancer cell lines, in contrast to their normal counterparts. We then demonstrated that knockdown of ANCR induced an EMT program and promoted cell migration and invasion in MCF10A (epithelial cells), whereas ectopic expression of ANCR repressed breast cancer cells migration and invasion. Furthermore, we validated in a nude mouse model that overexpression of ANCR in highly malignant and invasive MDA-MB-231 breast cancer cells significantly reduced the ability of the cells to form tumors and prevented the lung metastasis in vivo. Based on these data, our findings define a new mechanism underlying modulation of EZH2 stability by linking ANCR interaction with EZH2 to promote its phosphorylation that facilitates EZH2 degradation and suppresses breast cancer progression.

Topics: Animals; Breast Neoplasms; Cadherins; CDC2 Protein Kinase; Cell Line, Tumor; Cell Movement; Cycloheximide; Enhancer of Zeste Homolog 2 Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Lung Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Proteasome Endopeptidase Complex; Protein Binding; RNA, Long Noncoding; Ubiquitin

Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bortezomib; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinases; Drug Resistance, Neoplasm; Drug Synergism; Estradiol; Estrogen Receptor Modulators; Female; Fulvestrant; Humans; Leupeptins; MCF-7 Cells; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Raloxifene Hydrochloride; Receptors, Estrogen; RNA, Messenger; Tamoxifen

Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca

Topics: Breast Neoplasms; Calcitriol; Cell Line, Tumor; Cell Survival; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Leupeptins; Neoplasm Proteins; Proteasome Inhibitors; Proteolysis; Receptors, Calcitriol; RNA, Messenger; RNA, Small Interfering

Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.

Topics: Breast Neoplasms; Cell Line; Cell Proliferation; Cysteine Proteinase Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Immunoblotting; Leupeptins; MCF-7 Cells; Nuclear Proteins; Proteasome Endopeptidase Complex; Proteolysis; Reverse Transcriptase Polymerase Chain Reaction; RGS Proteins; Ribonucleases; Signal Transduction; Transcription Factors; Ubiquitin

Salvia officinalis is used as a dietary supplement with diverse medicinal activity (e.g. antidiabetic and antiatherosclerotic effects). The plant also exerts profound cytotoxicity toward cancer cells. Here, we investigated possible modes of action to explain its activity toward drug-resistant tumor cells. Log10IC50 values of two constituents of S. officinalis (ursolic acid, pomolic acid) were correlated to the expression of ATP-binding cassette (ABC) transporters (P-glycoprotein/ABCB1/MDR1, MRP1/ABCC1, BCRP/ABCG2) and epidermal growth factor receptor (EGFR) or mutations in RAS oncogenes and the tumor suppressor gene TP53 of the NCI panel of cell lines. Gene expression profiles predicting sensitivity and resistance of tumor cells to these compounds were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Furthermore, the binding of both plant acids to key molecules of the NF-κB pathway (NF-κB, I-κB, NEMO) was analyzed by molecular docking. Neither expression nor mutation of ABC transporters, oncogenes, or tumor suppressor genes correlated with log10IC50 values for ursolic acid or pomolic acid. In microarray analyses, many genes involved in signal transduction processes correlated with cellular responsiveness to these compounds. Molecular docking indicated that the two plant acids strongly bound to target proteins of the NF-κB pathway with even lower free binding energies than the known NF-κB inhibitor MG-132. They interacted more strongly with DNA-bound NF-κB than free NF-κB, pointing to inhibition of DNA binding by these compounds. In conclusion, the lack of cross-resistance to classical drug resistance mechanisms (ABC-transporters, oncogenes, tumor suppressors) may indicate a promising role of the both plant acids for cancer chemotherapy. Genes involved in signal transduction may contribute to the sensitivity or resistance of tumor cells to ursolic and pomolic acids. Ursolic and pomolic acid may target different steps of the NF-κB pathway to inhibit NF-κB-mediated functions.

Topics: Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Transporters; Breast Neoplasms; Drug Resistance, Neoplasm; Genes, erbB-1; Humans; Leupeptins; Molecular Structure; NF-kappa B; Oleanolic Acid; Pharmacogenetics; Plants, Medicinal; Salvia officinalis; Signal Transduction; Triterpenes; Ursolic Acid

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is known to enhance the cytotoxicity of the anticancer drug doxorubicin. On the other hand, breast cancer MCF-7 cells acquire resistance to doxorubicin under hypoxic conditions. In this study, we investigated the effect of resveratrol on hypoxia-induced resistance to doxorubicin in MCF-7 cells. Resveratrol and its derivative 3,5-dihydroxy-4'-methoxy-trans-stilbene, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene, cancelled hypoxia-induced resistance to doxorubicin at a concentration of 10 μM. Carbonyl reductase 1 (CBR1) catalyzes the conversion of doxorubicin to its metabolite doxorubicinol, which is much less effective than doxorubicin. Hypoxia increased the expression of CBR1 at both mRNA and protein levels, and knockdown of CBR1 inhibited hypoxia-induced resistance to doxorubicin in MCF-7 cells. Knockdown of hypoxia-inducible factor (HIF)-1α repressed the hypoxia-induced expression of CBR1. Resveratrol repressed the expression of HIF-1α protein, but not HIF-1α mRNA, and decreased hypoxia-activated HIF-1 activity. Resveratrol repressed the hypoxia-induced expression of CBR1 at both mRNA and protein levels. Likewise, 3,5-dihydroxy-4'-methoxy-trans-stilbene decreased the hypoxia-induced expression of CBR1 protein, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene. Furthermore, resveratrol decreased the expression of HIF-1α protein even in the presence of the proteasome inhibitor MG132 in hypoxia. Theses results indicate that in MCF-7 cells, HIF-1α-increased CBR1 expression plays an important role in hypoxia-induced resistance to doxorubicin and that resveratrol and 3,5-dihydroxy-4'-methoxy-trans-stilbene decrease CBR1 expression by decreasing HIF-1α protein expression, perhaps through a proteasome-independent pathway, and consequently repress hypoxia-induced resistance to doxorubicin.

Topics: Alcohol Oxidoreductases; Antineoplastic Agents; Breast Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; MCF-7 Cells; Phytotherapy; Plant Extracts; Resveratrol; RNA, Messenger; Stilbenes

Hsp90 is an important driver of stabilization and activation of several oncogenic proteins in many key pathways in oncogenesis, including HER2. The present study demonstrated that synuclein gamma (SNCG) prevents the protein degradation and protects the function of HER2 in the condition when the function of Hsp90 is blocked. Disruption of Hsp90 resulted in a significant degradation of HER2 and the loss of activity. However, SNCG completely recovered Hsp90 disruption-mediated losses of HER2 and the function. SNCG bound to HER2 in the presence and absence of Hsp90. Specifically, the C-terminal (Gln106-Asp127) of SNCG bound to the loop connecting αC helix and β4 sheet of the kinase domain of HER2. SNCG renders resistance to 17-AAG-induced tumor suppression in tumor xenograft. Crossing SNCG transgenic mice with HER2 mice stimulated HER2-induced tumor growth and rendered resistance to Hsp90 disruption. The present study indicates that SNCG protects Hsp90 client protein of HER2, and renders resistance to Hsp90 disruption.

Topics: Animals; Benzoquinones; Breast Neoplasms; Cell Line, Tumor; gamma-Synuclein; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Leupeptins; Mice; Mice, Nude; Mice, Transgenic; Receptor, ErbB-2

The ubiquitin-proteasome system and the autophagy-lysosome pathway are the two main mechanisms for eukaryotic intracellular protein degradation. Proteasome inhibitors are used for the treatment of some types of cancer, whereas autophagy seems to have a dual role in tumor cell survival and death. However, the relationship between both pathways has not been extensively studied in tumor cells. We have investigated both proteolytic systems in the human epithelial breast non-tumor cell line MCF10A and in the human epithelial breast tumor cell line MCF7. In basal condition, tumor cells showed a lower proteasome function but a higher autophagy activity when compared with MCF10A cells. Importantly, proteasome inhibition (PI) leads to different responses in both cell types. Tumor cells showed a dose-dependent glycogen synthase kinase-3 (GSK-3)β inhibition, a huge increase in the expression of the transcription factor CHOP and an active processing of caspase-8. By contrast, MCF10A cells fully activated GSK-3β and showed a lower expression of both CHOP and processed caspase-8. These molecular differences were reflected in a dose-dependent autophagy activation and cell death in tumor cells, while non-tumor cells exhibited the formation of inclusion bodies and a decrease in the cell death rate. Importantly, the behavior of the MCF7 cells can be reproduced in MCF10A cells when GSK-3β and the proteasome were simultaneously inhibited. Under this situation, MCF10A cells strongly activated autophagy, showing minimal inclusion bodies, increased CHOP expression and cell death rate. These findings support GSK-3β signaling as a key mechanism in regulating autophagy activation or inclusion formation in human tumor or non-tumor breast cells, respectively, which may shed new light on breast cancer control.

Topics: Antineoplastic Agents; Autophagy; Breast Neoplasms; Caspase 8; Cell Line, Tumor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Inclusion Bodies; Leupeptins; Organ Specificity; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Signal Transduction; Transcription Factor CHOP; Ubiquitin

Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

Topics: Animals; Breast Neoplasms; cdc42 GTP-Binding Protein; Cell Proliferation; Endothelial Cells; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Leupeptins; MCF-7 Cells; Mice; Middle Aged; Neovascularization, Pathologic; Protein Stability; rac1 GTP-Binding Protein; Tumor Suppressor Protein p53; Ubiquitination; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Junctional adhesion molecule-A (JAM-A) is a membranous cell-cell adhesion protein involved in tight-junction formation in epithelial and endothelial cells. Its overexpression in breast tumors has recently been linked with increased risk of metastasis. We sought to identify if JAM-A overexpression was associated with specific subtypes of breast cancer as defined by the expression of human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor. To this end, JAM-A immunohistochemistry was performed in two breast cancer tissue microarrays. In parallel, cross-talk between JAM-A, HER2 and ER was examined in several breast cell lines, using complementary genetic and pharmacological approaches. High JAM-A expression correlated significantly with HER2 protein expression, ER negativity, lower patient age, high-grade breast cancers, and aggressive luminal B, HER2 and basal subtypes of breast cancer. JAM-A and HER2 were co-expressed at high levels in vitro in SKBR3, UACC-812, UACC-893 and MCF7-HER2 cells. Knockdown or functional antagonism of HER2 did not alter JAM-A expression in any cell line tested. Interestingly, however, JAM-A knockdown decreased HER2 and ER-α expression, resulting in reduced levels of phospho-(active) AKT without an effect on the extracellular signal-related kinase phosphorylation. The downstream effects of JAM-A knockdown on HER2 and phospho-AKT were partially reversed upon treatment with the proteasomal inhibitor MG132. We conclude that JAM-A is co-expressed with HER2 and associates with aggressive breast cancer phenotypes. Furthermore, we speculate that JAM-A may regulate HER2 proteasomal degradation and activity, potentially offering a promise as a therapeutic target in HER2-positive breast cancers.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Cell Adhesion Molecules; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; MCF-7 Cells; Middle Aged; Phosphorylation; Proteolysis; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Cell Surface; Receptors, Estrogen; Receptors, Progesterone; RNA Interference; RNA, Small Interfering; Signal Transduction; Tissue Array Analysis

Protein degradation by the proteasome plays an important role in all major cellular pathways. Aberrant proteasome activity is associated with numerous human diseases including cancer and neurological disorders, but the underlying mechanism is virtually unclear. At least part of the reason for this is due to lack of understanding of the regulation of human proteasome genes. In this study, we found that a large set of human proteasome genes carry the CCAAT box in their promoters. We further demonstrated that the basal expression of these CCAAT box-containing proteasome genes is regulated by the transcription factor NF-Y. Knockdown of NF-YA, an essential subunit of NF-Y, reduced proteasome gene expression and compromised the cellular proteasome activity. In addition, we showed that knockdown of NF-YA sensitized breast cancer cells to the proteasome inhibitor MG132. This study unveils a new role for NF-Y in the regulation of human proteasome genes and suggests that NF-Y may be a potential target for cancer therapy.

Topics: Base Sequence; Breast Neoplasms; CCAAT-Binding Factor; Cell Line; Drug Screening Assays, Antitumor; Female; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Leupeptins; Molecular Sequence Data; Phylogeny; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding

Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.

Topics: Apoptosis; Autophagy; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leupeptins; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proteasome Inhibitors; Ribosomal Protein S6 Kinases, 70-kDa

Heme oxygenase-1 (HO-1) is an antioxidant, antiapoptotic and cytoprotective enzyme, catalysing the degradation of heme to carbon monoxide, biliverdin and ferrous iron. Recent studies indicated that expression of HO-1 is under the control of proapoptotic transcription factor p53 and antioxidant transcription factor Nrf2. Whether each of these transcription factors act independently or there is a cooperation between them in inducing HO-1 expression remains to be elucidated. In this study, we examined the expression of HO-1 in B16F10 melanoma and 4T1 breast cancer cells after cell exposure to proteasome inhibitors. We found that HO-1 protein level is increased by about 70% in p53-wt B16F10 cells in response to proteasome inhibitor MG132 after 6 h. Likewise, a 6.8 fold increase in HO-1 level was observed after cell exposure to the highly specific proteasome inhibitor bortezomib after 6 h of treatment in B16F10 cells. Whereas no induction of HO-1 was observed in p53-null 4T1 cells after treatment with bortezomib for 6 h. Next, we aligned HO-1 untranslated region with a consensus p53-responsive element. This bioinformatic analysis identified a p53-responsive element within the untranslated region of HO-1. Then, we examined HO-1 expression after a prolonged exposure to bortezomib in both B16F10 and 4T1 cell. These analyses similarly indicated that HO-1 is strongly induced in B16F10 cells in a dosedependent; contrary to our expectations, a strong induction of HO-1 is also observed in 4T1 cells. Therefore, it is concluded that HO-1 expression is under the control of p53 during early time points of proteasomal inhibition. However, during prolonged incubation with proteasome inhibitors, HO-1 expression can be induced in a p53-independent manner, suggesting participation of other protein(s) with longer half-lives.

Topics: Animals; Base Sequence; Blotting, Western; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Induction; Female; Genes, p53; Heme Oxygenase-1; Humans; Leupeptins; Melanoma, Experimental; Mice; Molecular Sequence Data; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Regulatory Sequences, Nucleic Acid

Pristimerin isolated from Celastrus and Maytenus spp can inhibit proteasome activity. However, whether pristimerin can modulate cancer metastasis is unknown.. The impacts of pristimerin on the purified and intracellular chymotrypsin proteasomal activity, the levels of regulator of G protein signaling 4 (RGS 4) expression and breast cancer cell lamellipodia formation, and the migration and invasion were determined by enzymatic, Western blot, immunofluorescent, and transwell assays, respectively.. We found that pristimerin inhibited human chymotrypsin proteasomal activity in MDA-MB-231 cells in a dose-dependent manner. Pristimerin also inhibited breast cancer cell lamellipodia formation, migration, and invasion in vitro by up-regulating RGS4 expression. Thus, knockdown of RGS4 attenuated pristimerin-mediated inhibition of breast cancer cell migration and invasion. Furthermore, pristimerin inhibited growth and invasion of implanted breast tumors in mice.. Pristmerin inhibits proteasomal activity and increases the levels of RGS4, inhibiting the migration and invasion of breast cancer cells.

Topics: Actins; Analysis of Variance; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chymases; Dose-Response Relationship, Drug; Female; Humans; Leupeptins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Pentacyclic Triterpenes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Random Allocation; RGS Proteins; RNA Interference; Triterpenes; Tumor Burden; Up-Regulation

Histone deacetylase 6 (HDAC6) inhibition, recently, has been shown to promote the acetylation of heat-shock protein 90 (Hsp90) and disrupt its chaperone function. Her-2 oncoprotein is identified as a client protein of Hsp90. Therefore, in this study we examined the effect of carbamazepine, which could inhibit HDAC on Hsp90 acetylation and Her-2 stability. The results of this study demonstrate that while carbamazepine had no effect on the Her-2 mRNA level, it induced Her-2 protein degradation via the proteasome pathway by disrupting the chaperone function of Hsp90 in SK-BR-3 cells. Mechanistically, carbamazepine could enhance the acetylation of α-tubulin, indicating its inhibitory effect on HDAC6. Functionally, carbamazepine could synergize with trastuzumab or geldanamycin to promote Her-2 degradation and inhibit breast cancer cell proliferation. Thus, this study has potential clinical implications by providing a promising strategy to overcome the development of resistance against trastuzumab therapy for breast cancer.

Topics: Acetylation; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzoquinones; Breast Neoplasms; Carbamazepine; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Female; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Stability; Receptor, ErbB-2; RNA, Messenger; Time Factors; Trastuzumab; Tubulin

The dietary isothiocyanates (ITCs) exhibit strong chemopreventive activities for a variety of neoplasms including breast cancer. However, the molecular mechanisms underlying ITC function in breast cancer cells have not been well established. Here, we found that phenethyl isothiocyanate (PEITC) acted more potently than the 'pure' anti-oestrogen ICI 182,780 to inhibit the growth of oestrogen receptor (ER)(+) breast cancer MCF7 and H3396 cells and ER(-) MDA-MB-231 and SK-BR-3 cells. PEITC reduced the steady state levels of ER-alpha and its novel variant, ER-alpha36 in a dose-and time-dependent manner and inhibited oestrogen-induced activation of the mitogen activated protein kinase/ERK 1/2 signaling pathway. However, ICI 182,780 that is potent in destabilization of ER-alpha protein, failed to down-regulate ER-alpha36. Our results thus demonstrated that PEITC functions as a more potent ER-alpha'disruptor' than the well-known ICI 182,780 to abrogate ER-mediated mitogenic oestrogen signaling in breast cancer cells, which provides a molecular explanation for the strong growth inhibitory activity of ITCs in breast cancer cells, and a rational for further exploration of ITCs as chemopreventive agents for human mammary carcinogenesis.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Estradiol; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Fulvestrant; Genome, Human; Humans; Isothiocyanates; Leupeptins; Phosphorylation; Signal Transduction

We investigated the anticancer effect of EGCG treatment on a breast carcinoma cell line resistant to tamoxifen (MCF-7Tam cells). As there are no reports about the molecular mechanisms implicated in EGCG treatment of tamoxifen resistant breast carcinoma cells, we studied the effects of EGCG treatment on three plasma membrane proteins that are involved in the mechanism of drug-resistance: Multidrug Resistance Protein (MRP1), P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). EGCG treatment (10-100 microg/ml for 24-72 hours) caused cell growth inhibition and dose-dependent apoptosis: after 100 microg/ml EGCG treatment for 24 hours, Bax expression increased and Bcl2 expression decreased (p<0.05). Coherently, Annexin V-FITC apoptosis assay detected a significant increase in labelled cells (p<0.05). EGCG did not affect MRP1: in contrast, 100 microg/ml EGCG administration caused P-gp decrease to 53% of control cells (p<0.001) and this effect was not due to downregulation of P-gp gene expression. EGCG induced P-gp decrease even when MG132, a strong proteasome inhibitor, was given together with EGCG to MCF-7Tam cells. EGCG treatment also inhibited BCRP activity: mRNA transcription and protein level did not change after treatment, but mitoxantrone test demonstrated a strong inhibition of BCRP activity (p<0.001). In conclusion, the present results showed that EGCG could down-regulate the activity of two molecules that play a key role in drug metabolism and transport and that are highly expressed in tamoxifen resistant breast carcinoma cells. The interaction of EGCG and drugs used in the therapy of estrogen sensitive breast carcinoma ought to be subject of studies and the potential use of EGCG in drug-resistant diseases ought to be better considered.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; Breast Neoplasms; Camellia sinensis; Catechin; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression; Humans; Leupeptins; Mitoxantrone; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Plant Extracts; Protease Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tamoxifen

Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interference-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 8; CDC2 Protein Kinase; Cell Line; Cyclin B1; Cysteine Proteinase Inhibitors; fas Receptor; Female; Humans; Leupeptins; Recombinant Fusion Proteins; Signal Transduction

The mechanisms responsible for 17β-estradiol (E(2))-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor α (ERα) antagonists are not fully understood. We describe a new tool for dissecting ERα action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor α that does not compete with estrogen for binding to ERα. TPSF noncompetitively inhibits estrogen-dependent ERα-mediated gene expression with little inhibition of transcriptional activity by NF-κB or the androgen or glucocorticoid receptor. TPSF inhibits E(2)-ERα-mediated induction of the proteinase inhibitor 9 gene, which is activated by ERα binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ERα to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E(2)-ERα-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ERα-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ERαHA cells that overexpress ERα, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ERα protein levels in MCF-7 cells and several other cell lines without altering ERα mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ERα by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ERα. TPSF represents a novel class of ER inhibitor with significant clinical potential.

Topics: Breast Neoplasms; Butyrophenones; Cell Line, Tumor; Estrogen Receptor alpha; Female; Fluorescence Polarization; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Leupeptins; Models, Chemical; Mucin-1; Proteasome Endopeptidase Complex; Purines; Response Elements; RNA, Messenger; Tamoxifen

Sulforaphane [SUL, 1-isothiocyanato-4-(methylsulfinyl)butane] is an isothiocyanate derived from glucoraphanin present in cruciferous vegetables, and it has a variety of potential chemopreventive actions. We analyzed the effects of SUL on the proliferation of human breast cancer cells and on the expression of estrogen receptor alpha (ERalpha) protein and mRNA in MCF-7 cells. Sulforaphane inhibited cell proliferation with IC(50) values at 24 and 48 h of 12.5 and 7.5 muM doses, respectively, and decreased ERalpha protein expression at concentrations between 2.5 and 30 muM. Inhibition of ERalpha protein expression was also accompanied by decreased progesterone receptor expression. MCF-7 ERalpha mRNA expression was inhibited by SUL at a dose of 30 muM, but not at lower SUL concentrations. At SUL doses <30 muM, the SUL-induced suppression of ERalpha protein was reversed by preincubation with the proteasome inhibitor MG132 and was accompanied by an increase in protein levels of the 20S catalytic core subunit PSMB5. Therefore, SUL can inhibit the expression of ERalpha protein in MCF-7 cells in part by inhibition of ERalpha mRNA transcription as well as by a mechanism that may involve increased proteasome-mediated degradation. These data provide new insights into mechanisms by which SUL inhibits proliferation of and down-regulates hormone receptor expression in MCF-7 cells.

Topics: Breast; Breast Neoplasms; Cell Line; Cell Proliferation; Down-Regulation; Estrogen Receptor alpha; Humans; Isothiocyanates; Leupeptins; RNA, Messenger; Sulfoxides; Thiocyanates; Tumor Cells, Cultured

Hypoxia activates and degrades estrogen receptor alpha (ERalpha) in human breast cancer cells, which may play an important role in the development and progression of breast cancer. In this study, the synergistic effects of estrogen (E(2)) and hypoxia on ERalpha-mediated transactivation and ERalpha degradation were investigated. ERalpha-mediated transcriptional activity was synergistically increased by E(2) and hypoxia, as determined by the transient expression of ERalpha and ER-responsive reporter plasmids in HEK 293 cells. Twenty hours of E(2) and hypoxia treatment synergistically induced degradation of ERalpha by 95% via a proteasome-dependent pathway in MCF-7 cells. These results provide evidence that hypoxia may stimulate yet unknown factor(s), which can further stimulate ERalpha signal transduction pathways.

Topics: Anaerobiosis; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Down-Regulation; Estradiol; Estrogen Receptor alpha; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Signal Transduction; Transcription, Genetic

Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Female; Humans; Leupeptins; Propiophenones; Propylamines; Proteasome Endopeptidase Complex; Proteasome Inhibitors

The transcription factor p73, a member of the p53 family, mediates cell-cycle arrest and apoptosis in response to DNA damage-induced cellular stress, acting thus as a proapoptotic gene. Similar to p53, p73 activity is regulated by post-translational modification, including phosphorylation, acetylation and ubiquitylation. In C. elegans, the F-box protein FSN-1 controls germline apoptosis by regulating CEP-1, the single ancestral p53 family member. Here we report that FBXO45, the human ortholog of FSN-1, binds specifically to p73 triggering its proteasome-dependent degradation. Importantly, SCF(FBXO45) ubiquitylates p73 both in vivo and in vitro. Moreover, siRNA-mediated depletion of FBXO45 stabilizes p73 and concomitantly induces cell death in a p53-independent manner. All together, these results show that the orphan F-box protein FBXO45 regulates the stability of p73, highlighting a conserved pathway evolved from nematode to human by which the p53 members are regulated by an SCF-dependent mechanism.

Topics: Animals; Breast Neoplasms; Cell Death; Cell Line; Cell Line, Transformed; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; DNA-Binding Proteins; F-Box Proteins; Green Fluorescent Proteins; HeLa Cells; Hemagglutinins; Humans; Kidney; Leupeptins; Mutation; Neuroblastoma; Nuclear Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; RNA, Small Interfering; Substrate Specificity; Temperature; Transfection; Tumor Protein p73; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination

gamma-Secretase inhibitors are new anti-cancer agents targeting Notch signaling. Their specificity for Notch is as yet unclear. Han and colleagues investigated the effects of Z-LeuLeuNleu-CHO on growth of breast cancer cells. The results demonstrated a reduction in cell viability primarily via proteasome inhibition independent of Notch activity. Currently, gamma-secretase inhibitors in clinical trials are structurally distinct from Z-LeuLeuNleu-CHO. Their effects on the proteasome are yet to be determined. However, findings from Han and colleagues pose two critical questions: Is the level of proteasomal activity in breast tumors the driving force for growth? What does the Notch pathway contribute to this growth?

Topics: Amyloid Precursor Protein Secretases; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Leupeptins; Oligopeptides; Receptors, Notch

Hypoxia, which is intimately associated with the biology of breast carcinomas, modulates the level of estrogen receptor (ER) alpha expression and transactivation. We investigated the effect of blocking ER degradation on ERalpha-mediated transactivation under hypoxic conditions using the proteasome inhibitor MG132. Pretreatment with MG132 blocked hypoxia-induced degradation of ERalpha protein. Our data imply that ERalpha proteasomal inhibition is linked to receptor transactivation under hypoxia.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Estrogen Receptor alpha; Female; Humans; Hypoxia; Leupeptins; Proteasome Inhibitors; Transcriptional Activation

The effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.. Solid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.. Among the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.. The ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.

Topics: Analysis of Variance; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Flavonoids; Humans; Lapatinib; Leupeptins; Lignans; Olive Oil; Phenols; Phosphorylation; Plant Oils; Polyphenols; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Quinazolines; Receptor, ErbB-2; RNA, Small Interfering; Trastuzumab

Clinical trials with proteasome inhibitor Bortezomib (also named Velcade or PS-341) has shown promising results for some cancers. However, other types of cancers including breast cancer do not respond well to Bortezomib. To understand the cause of the drug resistance, we compared the regulation of proteasome expression and the sensitivity to proteasome inhibitors between human breast cancer cells and nontumorigenic mammary epithelial cells. We found that, while the endogenous expression level is much higher, the potential of feedback expression in response to proteasome inhibitors is much lower in the breast cancer cells. Furthermore, the breast cancer cells are much more resistant to proteasome inhibitors compared to the nontumorigenic mammary epithelial cells. Biochemical analysis showed that the pathway of Bortezomib-induced apoptosis is apparently defective in the breast cancer cells. Together, these results provide an explanation for the inefficacy of Bortezomib in the clinical trials for breast cancer patients. The likelihood of combination therapy with Bortezomib and other anti-cancer agents for breast cancer is also discussed.

Topics: Apoptosis; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Feedback, Physiological; Humans; Leupeptins; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; Temperature; Tetrazolium Salts; Thiazoles

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

Topics: Base Sequence; Breast Neoplasms; Cell Membrane; Dipeptides; Enzyme Inhibitors; ErbB Receptors; Humans; Leupeptins; Molecular Sequence Data; Neuregulins; Phosphorylation; Protein Isoforms; Receptor, ErbB-4; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDACi) are a new class of anticancer agents that cause growth arrest, differentiation and/or apoptosis in many tumor cells. As acetylation regulates the activity of the anti-apoptotic transcription factor NF-kappaB, we investigated whether the proteasome inhibitor MG-132 would inhibit NF-kappaB activation and as a consequence potentiate HDACi-dependent apoptosis in breast cancer cells. We observed that the HDACi suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA) induced cell death but also enhanced NF-kappaB-activity. This increase of NF-kappaB activity was strongly reduced by the addition of MG-132. Moreover, MG-132 potentiates the HDACi-induced cell death that was associated with caspase-3 activation, and PARP cleavage. Induction of the stress related kinases JNK and p38 and the up-regulation of p21 and p27 were also observed after co-treatment of cells with HDACi and MG-132. Disruption of the NF-kappaB pathway by BAY 11-7085 or IkappaB-SR mimicked the action of MG-132 in promoting HDACi-induced cell death. Thus, the combined treatment with HDACi and proteasome inhibitors potentiates apoptosis in breast cancer cells representing a novel strategy for breast cancer therapy.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Colony-Forming Units Assay; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; I-kappa B Proteins; Leupeptins; Luciferases; Membrane Potential, Mitochondrial; NF-kappa B; NF-KappaB Inhibitor alpha; Nitriles; Sulfones; Transfection; Tumor Cells, Cultured; Vorinostat

Estrogen-induced proliferation in estrogen receptor (ER)-positive breast cancer cells is primarily mediated through two distinct intracellular receptors, ER alpha and ER beta. Although tumor necrosis factor alpha (TNF alpha) and E2/ER alpha are known to exert opposing effects on cell proliferation in MCF-7 cells, the mechanism by which TNFalpha antagonizes E2/ER alpha-mediated cell proliferation is not well understood. The present study suggests that reduced cell survival in response to TNF alpha treatment in MCF-7 cells may be associated with the down-regulation of ER alpha protein. The decrease in ER alpha protein level was accompanied by an inhibition of ER alpha gene transcription. Cell viability was decreased synergistically by the combined treatment with ER alpha-siRNA and TNF alpha. Furthermore, pretreatment of cells with the PI3-kinase (PI3K)/Akt inhibitor, LY294002, markedly enhanced TNF alpha-induced down-regulation of the ER alpha protein, suggesting that the PI3K/Akt pathway might be involved in control of the ER alpha level. Moreover, down-regulation of ER alpha by TNF alpha was not inhibited in cells that were pretreated with the proteasome inhibitors, MG132 and MG152, which suggests that proteasome-dependent proteolysis does not significantly influence TNF alpha-induced down-regulation of ER alpha protein. In contrast, the effect of the PI3K/Akt inhibitor on ER alpha was blocked in cells that were treated with LY294002 in the presence of the proteasome inhibitors. Collectively, our findings show that the TNF alpha may partly regulate the growth of MCF-7 breast cancer cells through the down-regulation of ER alpha expression, which is primarily mediated by a PI3K/Akt signaling.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Activation; Estrogen Receptor alpha; Female; Humans; Leupeptins; Phosphatidylinositol 3-Kinases; Proteasome Inhibitors; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

In mammalian cells, the level of estrogen receptor alpha (ERalpha) is rapidly decreased upon estrogen treatment, and this regulation involves proteasome degradation. Using different approaches, we showed that the Mdm2 oncogenic ubiquitin-ligase directly interacts with ERalpha in a ternary complex with p53 and is involved in the regulation of ERalpha turnover (both in the absence or presence of estrogens). Several lines of evidence indicated that this effect of Mdm2 required its ubiquitin-ligase activity and involved the ubiquitin/proteasome pathway. Moreover, in MCF-7 human breast cancer cells, various p53-inducing agents (such as UV irradiation) or treatment with RITA (which inhibits the interaction of p53 with Mdm2) stabilized ERalpha and abolished its 17beta-estradiol-dependent turnover. Interestingly, our data indicated that ligand-dependent receptor turnover was not required for efficient transactivation. Altogether, our results indicate that the Mdm2 oncoprotein and stress-inducing agents complexly and differentially regulate ERalpha stability and transcriptional activity in human cancer cells.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Estradiol; Estrogen Receptor alpha; HeLa Cells; Humans; Immunoprecipitation; Leupeptins; Neoplasm Proteins; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Transcriptional Activation; Tumor Suppressor Protein p53; Ultraviolet Rays

Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.

Topics: Blotting, Western; Breast Neoplasms; Cell Line; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Ecdysterone; Female; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; GTP-Binding Proteins; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Transgenes; Tumor Suppressor Proteins

Although ascorbate (Vitamin C) has been shown to inhibit cell growth and induce cell death in variety of cancer cells, results reported in other studies are inconsistent with this conclusion. It was previously reported that ascorbate induces apoptosis in human breast cancer cells. However, the molecular mechanism for this is not clear. In this study, we demonstrate that ascorbate induces cell death through the apoptosis-inducing factor (AIF) in the human breast cancer cell lines, SK-BR3 and Hs578T, but not in a normal breast cell line, Hs578. Ascorbate treatment caused the nuclear translocation of AIF, which is retained in the mitochondria in healthy cells, but caspase cleavage is not induced. Moreover, MG132, an inhibitor of AIF release from mitochondria, blocked the induction of cell death. Furthermore, cells that had been treated with human AIF-specific siRNA resisted cell death induced by ascorbate, implying that the translocation of AIF from mitochondria to the nucleus is responsible for ascorbate-mediated cell death. Therefore, these results suggest that ascorbate activates a caspase-independent and AIF-mediated cell death pathway in human breast cancer cells, SK-BR3, and Hs578T.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Apoptosis Inducing Factor; Ascorbic Acid; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Nucleus; Fluorescent Antibody Technique; Humans; Immunoblotting; Leupeptins; Mitochondria; Protein Transport; RNA, Small Interfering; Tumor Cells, Cultured

Although the anti-tumour effects of paclitaxel result mainly from mitotic arrest, recent evidences suggest alternative mechanisms of cytotoxicity. Cell cycle, cell death, and gene expression assays were used to understand the molecular mechanisms of paclitaxel cytotoxicity in breast cancer cells. G(2)/M cell cycle arrest and cell death coincided with the regulation of genes involved in cell death, cell cycle control, microtubule-based processes, oxidative stress, and ubiquitin-proteasome system. Induction of proteasome genes was also correlated with an accumulation of protein for proteasome subunits. Furthermore, a schedule-dependent regulation of paclitaxel-induced cytotoxicity was observed after combining paclitaxel and the proteasome inhibitor MG132. Proteasome inhibition after paclitaxel exposure induced the highest rate of growth inhibition and apoptosis, with no effect on mitotic arrest. These findings give support to clinical combinations of taxanes with proteasome inhibitors, outlining the importance of considering the sequence when designing such regimens.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Death; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Paclitaxel; Proteasome Endopeptidase Complex; Proteasome Inhibitors

1,1-Bis(3'-indolyl)methane (DIM) and the 5,5'-dibromo ring substituted DIM (5,5'-diBrDIM) inhibited growth of MCF-7 and MDA-MB-231 breast cancer cells, and IC50 values were 10-20 and 1-5 microM, respectively, in both cell lines. DIM and 5,5'-diBrDIM did not induce p21 or p27 protein levels or alter expression of Sp1 or Sp3 proteins in either cell line. In contrast, 10 microM 5,5'-diBrDIM downregulated cyclin D1 protein in MCF-7 and MDA-MB-231 cells 12 and 24 h after treatment. DIM (20 microM) also decreased cyclin D1 in MCF-7 (24 h) and MDA-MB-231 (12 h), and the DIM/5,5'-diBrDIM-induced degradation of cyclin D1 was blocked by the proteasome inhibitor MG132. Both DIM and 5,5'-diBrDIM induced apoptosis in MCF-7 cells and this was accompanied by decreased Bcl-2, release of mitochondrial cytochrome c, and decreased mitochondrial membrane potential as determined by the red/green fluorescence of JC-1. DIM and 5,5'-diBrDIM induced extensive necrosis in MDA-MB-231 cells; however, this was accompanied by decreased mitochondrial membrane potential primarily in cells treated with 5,5'-diBrDIM but not DIM. Thus, DIM and 5,5'-diBrDIM induce cell death in MCF-7 and MDA-MB-231 cells by overlapping and different pathways, and the ring-substituted DIM represents a novel class of uncharged mitochondrial poisons that inhibit breast cancer cell and tumor growth.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Cytochromes c; Female; Humans; Indoles; Inhibitory Concentration 50; Leupeptins; Mitochondria; Necrosis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Time Factors

HER-2/neu proto-oncogene is overexpressed in about one fourth of human breast cancers. AP-2 transcription factors bind to the HER-2/neu gene promoter and activate its expression. In a striking concurrence, anomalous abundance of AP-2alpha protein or its homolog AP-2gamma is also detected with HER-2/neu protein in mammary tumor-derived cell lines. This suggests that the deregulation of AP-2 is the preceding pathogenic event and probably the pivotal one in this type of mammary carcinogenesis. We examined the process of AP-2alpha gene expression in mammary carcinoma cell lines to identify where the aberration had occurred. We found no amplification of the AP-2alpha gene. Its promoter was marginally upregulated; however, it did not significantly increase the mRNA levels. When the AP-2alpha protein was examined, a remarkable stability was seen in breast cancer cell lines MDA-MB-453 and SK-BR-3, with a half-life of over 30 hr. This is sharply higher than the approximate 1 hr observed in mammary epithelial cell line MCF-10A and murine cell line NIH 3T3. Treatment of MCF-10A and NIH 3T3 cells with the proteasome inhibitor MG-132 showed that AP-2alpha was ubiquitinated and its level significantly increased. Moreover, this increase was accompanied by elevated levels HER-2/neu protein. In contrast, weaker ubiquitination of AP-2alpha was seen in MDA-MB-453 and SK-BR-3 cancer cells, and MG-132 treatment did not raise the AP-2alpha level any further. These results uncover that unusual stability is the main mechanism that raises the levels of AP-2 proteins, and in addition, provide the first clue that defective ubiquitin-dependent proteasomal-degradation pathway is possibly the prime cause that affects the HER-2/neu gene and culminates in breast cancer.

Topics: Animals; Breast Neoplasms; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Female; Gene Amplification; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Mammary Neoplasms, Animal; Mice; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proto-Oncogene Mas; Receptor, ErbB-2; Transcription Factor AP-2; Tumor Cells, Cultured; Ubiquitin; Up-Regulation

The proteasome inhibitor bortezomib is emerging as a potent anti-cancer agent. Still, recent clinical trials have revealed a significant secondary toxicity of bortezomib. Consequently, there is much interest in dissecting the mechanism of action of this compound to rationally improve its therapeutic index. The cytotoxic effect of bortezomib is frequently characterized by interfering with downstream events derived from the accumulation of proteasomal targets. Here we identify the first chemical agent able to act upstream of the proteasome to prevent cell killing by bortezomib. Specifically, we show that the polyhydroxyl compound Tiron can function as a competitive inhibitor of bortezomib. This effect of Tiron was surprising, since it is a classical radical spin trap and was expected to scavenge reactive oxygen species produced as a consequence of bortezomib action. The inhibitory effect of Tiron against bortezomib was selective, since it was not shared by other antioxidants, such as vitamin E, MnTBAP, L-N-acetyl-cysteine, and FK-506. Comparative analyses with nonboronated proteasome inhibitors (i.e. MG132) revealed a specificity of Tiron for bortezomib. We exploited this novel feature of Tiron to define the "point of no return" of proteasome inhibition in melanoma cells and to block cell death in a three-dimensional model of human skin. Cells from T-cell lymphoma, breast carcinoma, and non-small cell lung cancer were also responsive to Tiron, suggesting a broad impact of this agent as a bortezomib blocker. These results may have important implications for the analysis of bortezomib in vivo and for the design of drug mixtures containing proteasome inhibitors.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Antineoplastic Agents; Antioxidants; Binding, Competitive; Boronic Acids; Bortezomib; Breast Neoplasms; Cell Death; Cell Line; Cell Line, Tumor; Cell Survival; Drug Antagonism; Fibroblasts; Free Radicals; Humans; Immunoblotting; Kinetics; Leupeptins; Melanocytes; Melanoma; Membrane Potentials; Models, Biological; Protease Inhibitors; Proteasome Inhibitors; Pyrazines; Reactive Oxygen Species; Skin; Time Factors

Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.

Topics: Animals; Benzimidazoles; Blotting, Western; Breast Neoplasms; Calmodulin; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Electrophoresis, Polyacrylamide Gel; Gene Deletion; Humans; Immunoprecipitation; Leupeptins; Mutation; Point Mutation; Protease Inhibitors; Proteasome Endopeptidase Complex; Receptors, Estrogen; Transfection; Ubiquitin; Ubiquitin-Protein Ligases

Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3beta phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA) induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1) transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus.. Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3beta-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3beta/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells.. We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3beta-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

Topics: Acetylcysteine; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; Enzyme Inhibitors; Exportin 1 Protein; Fatty Acids, Unsaturated; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Karyopherins; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; RNA Interference; Transfection; Ubiquitin

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Luciferases; Male; Mice; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPkappa is cleaved by three proteases: furin, ADAM 10, and gamma-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPkappa. This is followed by gamma-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates beta-catenin, an RPTPkappa substrate. The expression of RPTPkappa suppresses beta-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of beta-catenin signaling. We also found that RPTPmu, another MAM family member, undergoes gamma-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPkappa signaling and implicate PIC in the activation of beta-catenin-mediated transcription.

Topics: ADAM Proteins; ADAM10 Protein; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; beta Catenin; Biotinylation; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cysteine Proteinase Inhibitors; Densitometry; Dose-Response Relationship, Drug; Endopeptidases; Female; Furin; Genes, Reporter; Green Fluorescent Proteins; HCT116 Cells; Humans; Kinetics; Leupeptins; Luciferases; Membrane Proteins; Mice; Models, Biological; NIH 3T3 Cells; Plasmids; Precipitin Tests; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 2; RNA Interference; Transcription, Genetic; Trifluoperazine

Induction of mRNA for BIK proapoptotic protein by doxorubicin or gamma-irradiation requires the DNA-binding transcription factor activity of p53. In MCF7 cells, pure antiestrogen fulvestrant also induces BIK mRNA and apoptosis. Here, we provide evidence that, in contrast to doxorubicin or gamma-irradiation, fulvestrant induction of BIK mRNA is not a direct effect of the transcriptional activity of p53, although p53 is necessary for this induction. It is known that p53 up-regulated modulator of apoptosis (PUMA) mRNA is induced directly by the transcriptional activity of p53. Whereas gamma-irradiation induced both BIK and PUMA mRNA, only BIK mRNA was induced by fulvestrant. Whereas both fulvestrant and doxorubicin induced BIK mRNA, only doxorubicin enhanced the DNA-binding activity of p53 and induced PUMA mRNA. Small interfering RNA (siRNA) suppression of p53 expression as well as overexpression of dominant-negative p53 effectively inhibited the fulvestrant induction of BIK mRNA, protein, and apoptosis. Transcriptional activity of a 2-kb BIK promoter, which contained an incomplete p53-binding sequence, was not affected by fulvestrant when tested by reporter assay. Fulvestrant neither affected the stability of the BIK mRNA transcripts. Interestingly, other human breast cancer cells, such as ZR75-1, constitutively expressed BIK mRNA even without fulvestrant. In these cells, however, BIK protein seemed to be rapidly degraded by proteasome, and siRNA suppression of BIK in ZR75-1 cells inhibited apoptosis induced by MG132 proteasome inhibitor. These results suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a nontranscriptional activity of p53 and by proteasomal degradation of BIK protein.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Estradiol; Fulvestrant; Gamma Rays; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Membrane Proteins; Mitochondrial Proteins; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Estrogen; RNA, Messenger; Transcription, Genetic; Tumor Suppressor Protein p53

The mammalian target of rapamycin is a serine-threonine kinase that regulates cell cycle progression. Rapamycin and its analogues inhibit the mammalian target of rapamycin and are being actively investigated in clinical trials as novel targeted anticancer agents. Although cyclin D1 is down-regulated by rapamycin, the role of this down-regulation in rapamycin-mediated growth inhibition and the mechanism of cyclin D1 down-regulation are not well understood. Here, we show that overexpression of cyclin D1 partially overcomes rapamycin-induced cell cycle arrest and inhibition of anchorage-dependent growth in breast cancer cells. Rapamycin not only decreases endogenous cyclin D1 levels but also decreases the expression of transfected cyclin D1, suggesting that this is at least in part caused by accelerated proteolysis. Indeed, rapamycin decreases the half-life of cyclin D1 protein, and the rapamycin-induced decrease in cyclin D1 levels is partially abrogated by proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Rapamycin treatment leads to an increase in the kinase activity of glycogen synthase kinase 3beta (GSK3beta), a known regulator of cyclin D1 proteolysis. Rapamycin-induced down-regulation of cyclin D1 is inhibited by the GSK3beta inhibitors lithium chloride, SB216763, and SB415286. Rapamycin-induced G1 arrest is abrogated by nonspecific GSK3beta inhibitor lithium chloride but not by selective inhibitor SB216763, suggesting that GSK3beta is not essential for rapamycin-mediated G1 arrest. However, rapamycin inhibits cell growth significantly more in GSK3beta wild-type cells than in GSK3beta-null cells, suggesting that GSK3beta enhances rapamycin-mediated growth inhibition. In addition, rapamycin enhances paclitaxel-induced apoptosis through the mitochondrial death pathway; this is inhibited by selective GSK3beta inhibitors SB216763 and SB415286. Furthermore, rapamycin significantly enhances paclitaxel-induced cytotoxicity in GSK3beta wild-type but not in GSK3beta-null cells, suggesting a critical role for GSK3beta in rapamycin-mediated paclitaxel-sensitization. Taken together, these results show that GSK3beta plays an important role in rapamycin-mediated cell cycle regulation and chemosensitivity and thus significantly potentiates the antitumor effects of rapamycin.

Topics: Aminophenols; Antibiotics, Antineoplastic; Antimanic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cyclin D1; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Resistance, Neoplasm; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Half-Life; Humans; Indoles; Leupeptins; Lithium Chloride; Maleimides; Mitochondria; NF-kappa B; Paclitaxel; Proteasome Inhibitors; Sirolimus

Estrogen receptor alpha (ER) turnover in MCF-7 cells was assessed by pulse chase analysis and measurement of ER steady-state level. In untreated cells, degradation of (35)S-labeled ER was characterized by a slow phase followed by a more rapid decline. Without ligand, ER elimination was totally compensated by synthesis which maintained receptor homeostasis. Estradiol (E(2)) and the pure antiestrogen RU 58,668 abolished the slow phase of ER breakdown and enhanced the degradation of neosynthesized ER, producing a low ER steady-state level. By contrast, the partial antiestrogen OH-Tam was ineffective in this respect and caused ER accumulation. Regardless of the conditions, ER breakdown was abolished by proteasome inhibition (MG-132). ER ligands decreased cell capacity to bind [(3)H]E(2), even in the presence of MG-132, indicating that the regulation of ER level and E(2) binding capacity occurs through distinct mechanisms. MG-132 partially blocked the basal transcription of an ERE-dependent reporter gene and modified the ability of E(2) to induce the expression of the latter: the hormone was unable to restore the transactivation activity measured without MG-132. RU 58,668 and OH-Tam failed to enhance the inhibitory action of MG-132, suggesting that a loss of basal ER-mediated transactivation mainly affects the stimulatory effect of estrogens. Overall, our findings reveal that ER steady state level, ligand binding capacity and transactivation potency fit in a complex regulatory scheme involving distinct mechanisms, which may be dissociated from each other under various treatments.

Topics: Breast Neoplasms; Cell Line, Tumor; Cycloheximide; Down-Regulation; Enzyme Inhibitors; Estradiol; Estrogen Receptor alpha; Genes, Reporter; Humans; Leupeptins; Ligands; Luciferases; Methionine; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sulfur Radioisotopes; Tamoxifen; Transcription, Genetic; Tritium

I have postulated that arachidonic acid release from rat liver cells is associated with cancer chemoprevention. Since it has been reported that inhibition of proteasome activities may prevent cancer, the effects of proteasome inhibitors on arachidonic acid release from cells and on prostaglandin I2 production in rat liver cells were studied.. The proteasome inhibitors, epoxomicin, lactacystin and carbobenzoxy-leucyl-leucyl-leucinal, stimulate the release of arachidonic acid from rat glial, human colon carcinoma, human breast carcinoma and the rat liver cells. They also stimulate basal and induced prostacycin production in the rat liver cells. The stimulated arachidonic acid release and basal prostaglandin I2 production in rat liver cells is inhibited by actinomycin D.. Stimulation of arachidonic acid release and arachidonic acid metabolism may be associated with some of the biologic effects observed after proteasome inhibition, e.g. prevention of tumor growth, induction of apoptosis, stimulation of bone formation.

Topics: Acetylcysteine; Animals; Arachidonic Acid; Breast Neoplasms; Cell Line; Cell Line, Tumor; HT29 Cells; Humans; Leupeptins; Liver; Neuroglia; Oligopeptides; Proteasome Inhibitors; Rats

The phenotypic changes of increased motility and invasiveness of cancer cells are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development. Snail, a zinc-finger transcription factor, triggers this process by repressing E-cadherin expression; however, the mechanisms that regulate Snail remain elusive. Here we find that Snail is highly unstable, with a short half-life about 25 min. We show that GSK-3beta binds to and phosphorylates Snail at two consensus motifs to dually regulate the function of this protein. Phosphorylation of the first motif regulates its beta-Trcp-mediated ubiquitination, whereas phosphorylation of the second motif controls its subcellular localization. A variant of Snail (Snail-6SA), which abolishes these phosphorylations, is much more stable and resides exclusively in the nucleus to induce EMT. Furthermore, inhibition of GSK-3beta results in the upregulation of Snail and downregulation of E-cadherin in vivo. Thus, Snail and GSK-3beta together function as a molecular switch for many signalling pathways that lead to EMT.

Topics: Amino Acid Motifs; Binding Sites; Breast Neoplasms; Cadherins; Cell Line, Tumor; Cell Nucleus; Consensus Sequence; Cysteine Endopeptidases; DNA-Binding Proteins; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Leupeptins; Lithium; Mesoderm; Multienzyme Complexes; Mutation; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Snail Family Transcription Factors; Substrate Specificity; Transcription Factors; Zinc Fingers

Molecular chaperones and co-chaperones, such as heat-shock proteins (Hsp's), play a pivotal role in the adequate folding and the stability of steroid hormone receptors. As shown by immunofluorescence staining and immunoblot analysis, the Hsp90 inhibitor radicicol induced a rapid (within hours) depletion of estrogen receptor-alpha (ER) in MCF-7 and IBEP-2 breast carcinoma cells. Inhibition of proteasomes (MG-132, LLnL) or of protein synthesis (cycloheximide), which both suppressed E(2)-induced downregulation of ER, failed to modify ER degradation caused by radicicol. On the other hand, partial antiestrogens, such as hydroxytamoxifen (a triphenylethylene) and LY 117,018 (a benzothiophene) stabilized ER, making it immune to radicicol-induced degradation. Furthermore, radicicol did not interfere with ER upregulation induced by hydroxytamoxifen. Thus, the current study points to possible variation in the mechanism/pathway of ER breakdown. Besides, the protective effect of partial antiestrogens suggests that ER stability is only compromized by Hsp90 disruption when the receptor is in its native, unliganded form.

Topics: Breast Neoplasms; Cycloheximide; Enzyme Inhibitors; Estrogen Antagonists; Estrogen Receptor alpha; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Lactones; Leupeptins; Ligands; Macrolides; Molecular Chaperones; Proteasome Inhibitors; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Pyrrolidines; Signal Transduction; Tamoxifen; Thiophenes; Tumor Cells, Cultured

Estrogen receptor alpha (ERalpha)-positive breast cancer cell lines are up to 10 times more sensitive than ERalpha-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response.. In the ERalpha-positive MCF-7 cell line, TSA repressed ERalpha and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G(1)-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERalpha-negative MDA-MB-231 cell line, which arrested in G(2)-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA.. Tamoxifen has been shown to inhibit ERalpha-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERalpha-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERalpha-dependent and ERalpha-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Endopeptidases; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; RNA Interference; S-Phase Kinase-Associated Proteins; Tamoxifen; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms

Normal mammary epithelial cells are rapidly induced to G(1) arrest by the widely expressed cytokine, transforming growth factor beta (TGF-beta1). Studies in established breast cancer cell lines that express the estrogen receptor alpha (ERalpha) have demonstrated loss of this responsiveness. This inverse correlation suggests interpathway signaling important to cell growth and regulation. The adenocarcinoma breast cell line BT474, which was not growth arrested by TGF-beta1, was used as a model of estrogen-inducible growth to explore interpathway crosstalk. Although BT474 cells were not growth-arrested by TGF-beta1 as determined by flow cytometry analysis and 5'-bromo-3'-deoxyuridine incorporation into DNA, estrogen receptor protein levels were attenuated by 100 pM TGF-beta1 after 6 h. This decrease in ERalpha reached 50% of untreated control levels by 24 h of treatment and was further supported by a 50% decrease in estrogen-inducible DNA synthesis. Inspection of ERalpha transcripts suggested that this decrease was primarily the result of altered ERalpha protein stability or availability. Use of the proteasome inhibitor, MG132, abolished all effects on ERalpha by TGF-beta1. Collectively, this data supports a role for TGF-beta1 in regulating the growth of otherwise insensitive breast cancer cells through modulation of ERalpha stability.

Topics: Blotting, Northern; Blotting, Western; Breast Neoplasms; Bromodeoxyuridine; Cell Division; Cysteine Endopeptidases; Estradiol; Estrogen Receptor alpha; Flow Cytometry; Humans; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Ubiquitin

p62 is a multifunctional cytoplasmic protein able to noncovalently bind ubiquitin and several signaling proteins, suggesting a regulatory role connected to the ubiquitin-proteasome pathway. No studies to date have linked p62 protein expression with pathological states. Here we demonstrate the overabundance of p62 protein in malignant breast tissue relative to normal breast tissue. The proteasome inhibitor PSI increased p62 mRNA and protein; however, PSI treatment of breast epithelial cells transfected with the p62 promoter did not affect promoter activity. High levels of prostate-derived Ets factor (PDEF) mRNA have been identified in breast cancer compared to normal breast. Only the PSA and maspin promoters have been identified as targets of this transcription factor. Here we show that PDEF stimulates the p62 promoter through at least two sites, and likely acts as a coactivator. PSI treatment abrogates the PDEF-stimulated increase of p62 promoter activity by 50%. Thus, multiple mechanisms for the induction of p62 exist. We conclude that (1) p62 protein is overexpressed in breast cancer; (2) p62 mRNA and protein increase in response to PSI, with no change of basal promoter activity; (3) PDEF upregulates p62 promoter activity through at least two sites; and (4) PSI downregulates PDEF-induced p62 promoter activation through one of these sites.

Topics: Acetylcysteine; Adaptor Proteins, Signal Transducing; Breast; Breast Neoplasms; Carrier Proteins; Cells, Cultured; Computer Systems; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Oligopeptides; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteins; Proto-Oncogene Proteins c-ets; Regulatory Sequences, Nucleic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sequestosome-1 Protein; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Ubiquitin

Estrogen receptor alpha (ER alpha) degradation is regulated by ubiquitination, but the signaling pathways that modulate ER alpha turnover are unknown. We found that extracellular signal-regulated kinase 7 (ERK7) preferentially enhances the destruction of ER alpha but not the related androgen receptor. Loss of ERK7 was correlated with breast cancer progression, and all ER alpha-positive breast tumors had decreased ERK7 expression compared to that found in normal breast tissue. In human breast cells, a dominant-negative ERK7 mutant decreased the rate of endogenous ER alpha degradation >4-fold in the presence of hormone and potentiated estrogen responsiveness. ERK7 targets the ER alpha ligand-binding domain for destruction by enhancing its ubiquitination. Thus, ERK7 is a novel regulator of estrogen responsiveness through its control of ER alpha turnover.

Topics: Animals; Binding Sites; Breast; Breast Neoplasms; Cells, Cultured; Cricetinae; Cysteine Proteinase Inhibitors; Estrogen Receptor alpha; Extracellular Signal-Regulated MAP Kinases; Female; Hormones; Humans; Kidney; Leupeptins; Mitogen-Activated Protein Kinases; Mutation; Peptide Hydrolases; Phosphorylation; Proteasome Endopeptidase Complex; Receptors, Estrogen; Reference Values; Signal Transduction; Ubiquitin

SHP-1 has been proposed to be a tumor suppressor gene for several cancers. The expression of SHP-1 protein is diminished or abolished in most leukemia and lymphoma cell lines and tissues, and in some non-hematopoietic cancer cell lines, such as estrogen receptor (ER) negative breast cancer cell lines and some colorectal cancer cell lines. However, we do not know whether the reduced SHP-1 expression is the cause of cancer diseases or the secondary effect of cancer developments. Here, we first demonstrate that SHP-1 has general tumor suppressing function in SHP-1 transfected cell lines. Transfected SHP-1 inhibits the growth of three lymphoma/leukemia cell lines (Ramos, H9, Jurkat) and one breast cancer cell line (HTB26). We also demonstrate a possible molecular mechanism for the tumor suppressing function of SHP-1: SHP-1 inhibits cell growth partly by negative regulation of activated JAK kinase. In addition, we find, for the first time, that SHP-1 down-regulates the level of TYK2 kinase in H9 cells and of JAK1 kinase in HTB26 cells, by accelerating their degradation. The SHP-1 accelerated degradation of JAK1 kinase in HTB26 cells was blocked with the treatment of MG132, a specific inhibitor for proteasome-mediated proteolysis. Our data suggest a new function of SHP-1 in the regulation of proteasome-mediated degradation pathway.

Topics: Breast Neoplasms; Cell Division; Cysteine Proteinase Inhibitors; Down-Regulation; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Janus Kinase 1; Leukemia; Leupeptins; Lymphoma; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; src Homology Domains; Transfection; Tumor Cells, Cultured; TYK2 Kinase

Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.

Topics: Breast Neoplasms; Cell Division; Cell Hypoxia; Cobalt; Cysteine Endopeptidases; Endothelial Growth Factors; Estradiol; Estrogen Receptor alpha; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Signaling Peptides and Proteins; Leupeptins; Lymphokines; Multienzyme Complexes; Promoter Regions, Genetic; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteins; Receptors, Estrogen; Response Elements; Sp1 Transcription Factor; Transcription Factors; Transcriptional Activation; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Full length Mcl-1 is an anti-apoptotic protein consisting of two closely migrating 42/40kDa species. We now investigated the relationship of these isoforms to the expression of cell cycle stimulatory (cyclin A) and inhibitory (p21WAF1) proteins and to the induction of apoptosis in wt p53 MCF-7 and mutant p53 SKBR3 human breast carcinomas. The latter cells exhibited lower 42kDa Mcl-1, higher expression of cyclin A relative to that of p21WAF1, and apoptosis in response to okadaic acid, a phosphatase 1/2A inhibitor. The proteasome inhibitor MG-115 selectively increased expression of the 40kDa Mcl-1 isoform and induced p21WAF1, but also promoted preferential apoptosis in SKBR3 cells. Neither okadaic acid nor MG-115 caused comparable effects in MCF-7 cells. However, vanadate or acetyl furanonaphthoquinone induced the 40kDa Mcl-1 and greater Jun kinase (JNK) phosphorylation without apoptosis-associated PARP fragmentation in MCF-7 cells. Our data suggest that the higher susceptibility of SKBR3 cells to undergo apoptosis may be partly due to their greater proliferative potential (cyclin A), low expression of the anti-apoptotic 42kDa Mcl-1 isoform, and suboptimal JNK activation in response to stress.

Topics: Apoptosis; Breast Neoplasms; Cell Nucleus; Chromatin; Cyclin A; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Female; Genes, p53; Humans; JNK Mitogen-Activated Protein Kinases; Leupeptins; Mitogen-Activated Protein Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Okadaic Acid; Phosphorylation; Protease Inhibitors; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Transcription Factors; Tumor Cells, Cultured

Growth-inhibitory effects on DS19 mouse erythroleukemia cells were seen in the micromolar concentration range with allicin and S-allylmercaptocysteine and in the millimolar range with allyl butyrate, allyl phenyl sulfone, and S-allyl cysteine. Increased acetylation of histones was induced by incubation of cells with the allyl compounds at concentrations similar to those that resulted in the inhibition of cell proliferation. The induction of histone acetylation by S-allylmercaptocysteine was also observed in Caco-2 human colon cancer cells and T47D human breast cancer cells. In contrast to the effect on histone acetylation, there was a decrease in the incorporation of phosphate into histones when DS19 cells were incubated with 25 microM S-allylmercaptocysteine. Histone deacetylase activity was inhibited by allyl butyrate, but there was little or no effect with the allyl sulfur compounds examined in this study. A similar degree of downregulation of histone deacetylase and histone acetyltransferase was observed when DS19 cells were incubated with S-allylmercaptocysteine or allyl isothiocyanate. The induction of histone acetylation by S-allylmercaptocysteine was not blocked by a proteasome inhibitor. The mechanism by which S-allylmercaptocysteine induces histone acetylation remains to be characterized. It may be related in part to metabolism to allyl mercaptan, which is a more effective inhibitor of histone deacetylase.

Topics: Acetylation; Acetyltransferases; Allyl Compounds; Animals; Antineoplastic Agents; Breast Neoplasms; Colonic Neoplasms; Cysteine; Disulfides; Electrophoresis, Polyacrylamide Gel; Female; Histone Acetyltransferases; Histone Deacetylases; Histones; Humans; Leukemia, Erythroblastic, Acute; Leupeptins; Mice; Saccharomyces cerevisiae Proteins; Sulfinic Acids; Tumor Cells, Cultured

The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues.

Topics: Animals; Antioxidants; Breast Neoplasms; Cells, Cultured; Chelating Agents; Copper; Cysteine; Cysteine Proteinase Inhibitors; Ditiocarb; Female; Fibroblasts; Free Radicals; Humans; Hydrogen Peroxide; Leupeptins; Mannitol; Molecular Structure; Oxidants; Oxidation-Reduction; Pyrrolidines; Rats; Recombinant Proteins; Thiobarbituric Acid Reactive Substances; Thiocarbamates; Tumor Suppressor Protein p53

The ubiquitin-proteasome pathway regulates the turnover of many transcription factors, including steroid hormone receptors such as the estrogen receptor and progesterone receptor. For these receptors, proteasome inhibition interferes with steroid-mediated transcription. We show here that proteasome inhibition with MG132 results in increased accumulation of the glucocorticoid receptor (GR), confirming that it is likewise a substrate for the ubiquitin-proteasome degradative pathway. Using the mouse mammary tumor virus (MMTV) promoter integrated into tissue culture cells, we found that proteasome inhibition synergistically increases GR-mediated transactivation. This increased activation was observed in a number of cell lines and on various MMTV templates, either as transiently transfected reporters or stably integrated into chromatin. These observations suggest that the increase in GR-mediated transcription due to proteasome inhibition may occur downstream of the initial chromatin remodeling step. In support of this concept, the increase in transcription did not correlate with an increase in chromatin remodeling, as measured by restriction enzyme hypersensitivity, or transcription factor loading, as exemplified by nuclear factor 1. To investigate the relationship between GR turnover, transcription, and subnuclear trafficking, we examined the effect of proteasome inhibition on the mobility of the GR within the nucleus and association of the GR with the nuclear matrix. Blocking GR turnover reduced the mobility of the GR within the nucleus, and this correlated with increased association of the receptor with the nuclear matrix. As a result of proteasome inhibition, GR mobility within the nucleus was reduced while its association with the nuclear matrix was increased. Thus, while altered nuclear mobility of steroid receptors may be a common feature of proteasome inhibition, GR is unique in its enhanced transactivation activity that results when proteasome function is compromised. Proteasomes may therefore impact steroid receptor action at multiple levels and exert distinct effects on individual receptor types.

Topics: Breast Neoplasms; Cell Nucleus; Chromatin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Helicases; DNA-Binding Proteins; Female; Histone Acetyltransferases; Humans; Leupeptins; Mammary Tumor Virus, Mouse; Multienzyme Complexes; Nuclear Proteins; Nuclear Receptor Coactivator 1; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Transport; Receptors, Glucocorticoid; Repressor Proteins; Sin3 Histone Deacetylase and Corepressor Complex; Templates, Genetic; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Most studies have reported an up-regulation of retinoic acid receptor (RAR) mRNA expression by all-trans retinoic acid (RA). We aimed to study the effect of RA on RAR protein levels in MCF-7 human breast cancer cells. Incubation of these cells with 10(-6) M RA induced a rapid breakdown of both RARalpha and RARgamma in spite of the accumulation of their mRNAs. Proteasome specific inhibitors blocked the RA-induced breakdown of RARs. Furthermore, RA enhanced the formation of the complex between RARalpha and ubiquitin in a concentration- and time-dependent manner, suggesting the involvement of ubiquitin and proteasome in this reaction. Retinoid X receptor alpha (RXRalpha) was also decreased, albeit to a lesser extent, in RA-treated cells. Use of synthetic receptor agonists and antagonists clearly showed that the effect of the retinoid on the breakdown of the retinoid receptors is receptor-ligand agonist-dependent and blunted by the antagonist. An electrophoretic mobility shift assay, using nuclear extracts from RA-treated cells, showed that a reduction in complex formation with hormone response elements correlated with the reduction of RAR and RXR protein. These data suggest that RA induces the breakdown of RARs through a process involving ubiquitination and that this phenomenon causes a reduction in the formation of DNA-receptor complexes.

Topics: Antineoplastic Agents; Breast Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA; Humans; Leupeptins; Multienzyme Complexes; Nuclear Proteins; Proteasome Endopeptidase Complex; Receptors, Retinoic Acid; Response Elements; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Tretinoin; Tumor Cells, Cultured; Ubiquitins

Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells. Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death. The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine. We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.

Topics: Acetylation; Acetylcysteine; Animals; Anti-Bacterial Agents; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Chickens; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeleton; Down-Regulation; Enzyme Inhibitors; Female; G1 Phase; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immunoblotting; Keratins; Leupeptins; Multienzyme Complexes; Peptides; Phosphorylation; Proteasome Endopeptidase Complex; Quinidine; Retinoblastoma Protein; Time Factors; Tumor Cells, Cultured

We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Apoptosis; Autoantigens; Blotting, Western; BRCA1 Protein; Breast Neoplasms; Calpain; Carrier Proteins; Cell Cycle; Cell Fractionation; Cloning, Molecular; Colonic Neoplasms; Cysteine Proteinase Inhibitors; DNA, Complementary; Egtazic Acid; Enzyme Inhibitors; Gene Library; Humans; Leupeptins; Mammary Neoplasms, Animal; Mice; Molecular Sequence Data; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Rats; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

The E6 proteins derived from tumour associated papillomavirus types target the cellular tumour suppressor protein p53 for ubiquitin mediated degradation. In cell lines derived from cervical tumours the p53 protein is present in very low amounts, but it can be activated by appropriate DNA damaging agents, indicating that functional p53 is present within these lines. Recent studies have also shown that different polymorphic forms of the p53 protein are differentially susceptible to E6 mediated degradation. Therefore we have been interested in analysing the effects of different HPV E6 proteins upon p53 levels in a variety of cervical tumour derived cell lines. We show that inhibition of E6 mediated degradation of p53 frequently results in increased levels of p53 expression. However, there are notable exceptions to this where increased p53 levels are only obtained following DNA damage and proteasome inhibition. We also show in E6 expressing cells, that as well as p53 being targeted for degradation, the localization of p53 to the nucleus is also inhibited, consistent with previous observations which indicate that degradation of p53 is not essential for E6 mediated inhibition of p53 function. These results have important implications for any potential therapies which might aim to block E6 mediated degradation of p53.

Topics: Acetylcysteine; Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Carcinoma; Cell Nucleus; Cysteine Proteinase Inhibitors; DNA Damage; DNA-Binding Proteins; Female; Fibrosarcoma; Humans; Leupeptins; Mitomycin; Oncogene Proteins, Viral; Papillomaviridae; Polymorphism, Genetic; Repressor Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.

Topics: Acetylcysteine; Breast Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; DNA Replication; Female; Histones; Hot Temperature; Humans; Leupeptins; Multienzyme Complexes; Nucleoproteins; Nucleosomes; Proteasome Endopeptidase Complex; Protein Biosynthesis; Transcription, Genetic; Tumor Cells, Cultured; Ubiquitins

A 52K glycoprotein is secreted by human breast cancer cells in culture after estrogen stimulation. Using monoclonal antibodies, we have quantitated and characterized the corresponding proteins of the cell compartment. Using pulse-chase experiments, we have shown that about 40% of the 52K protein is secreted, the majority being successively processed into a 48K and a 34K protein. This last protein is very stable. The processing is inhibited by lysosomotropic agents and leupeptin, suggesting that it occurs in acidic vesicles, such as lysosomes or endosomes. Estradiol increased the intracellular level of immunoreactive 52K related proteins by 4-fold. Its effect is, however, more obvious in the medium, since there is a constitutive level in the cell. The stimulatory effects of estradiol on [3H]mannose and [35S]methionine incorporation into these proteins were similar and the endoglycosydase H sensitivity of the proteins was not altered, suggesting that estradiol did not modulate the glycosylation step. Antiestrogens did not stimulate synthesis and glycosylation of the 52K related proteins. Estradiol also increased the stability of the 52K precursor as well as that of total proteins. We conclude that the secreted 52K protein is the precursor of two cellular proteins of 48K and 34K. Estradiol stimulates both the intracellular accumulation of these proteins and the secretion of the precursor.

Topics: Ammonium Chloride; Antibodies, Monoclonal; Breast Neoplasms; Cell Compartmentation; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation; Glycosylation; Humans; Leupeptins; Molecular Weight; Monensin; Neoplasm Proteins; Protein Processing, Post-Translational

Proteolytic activity in human breast cancer cytosols was studied using hormone receptors from rats as the substrates. Under the conditions tested, limited proteolysis of both the estrogen and the progesterone receptors in uterine cytosol was observed, but not proteolysis of the glucocorticoid or androgen receptors in liver or prostate cytosols, respectively. Although both the nonactivated and activated uterine estrogen receptors were attacked by the enzyme(s), molybdate-stabilized receptors were resistant to proteolysis. The product of estrogen receptor cleavage sedimented at approximately 4S in low-salt gradients and at 3 to 4S in high-salt gradients. This fragment retained both the steroid-binding and DNA-binding domains. The marked decrease in its DNA-binding ability, compared with the salt-dissociated but non-proteolyzed receptors, may be attributable to interactions of the fragment with dialyzable modulator(s) in cytosol. The proteolytic activity in tumor cytosol was leupeptin sensitive and was precipitated by (NH4)2SO4 at 30 to 60% saturation. Its sedimentation coefficient was 4 to 5S. The proteolytic activity was identified in 70% of estrogen receptor-negative tumors but in only 40% of estrogen receptor-positive tumors.

Topics: Breast Neoplasms; Cytosol; Estradiol; Female; Humans; Kinetics; Leupeptins; Molybdenum; Peptide Hydrolases; Receptors, Estradiol; Receptors, Estrogen; Receptors, Progesterone; Substrate Specificity; Uterus

Estrogen receptors from rat uterus and human breast carcinoma were analyzed by diethylaminoethyl-cellulose chromatography. Cytosols which had been incubated for short periods of time demonstrated a single discrete elution peak, indicating a single ionic form, while cytosols incubated for longer periods of time generated a second ionic form of receptor. Addition of cations to cytosols also promoted the rapid appearance of this second ionic form of receptor. Either leupeptin, a protease inhibitor, or sodium molybdate prevented the appearance of this second ionic form of estrogen receptor. The estrogen receptor from rat uterine cytosol incubated without leupeptin or molybdate had a smaller apparent molecular weight than did estrogen receptor from cytosols incubated with leupeptin or molybdate. Altogether, these experiments suggested that a cation-dependent protease present in the cytosols from both tissues was degrading the estrogen receptor to a second smaller species during extended incubation times.

Topics: Animals; Breast Neoplasms; Chromatography, DEAE-Cellulose; Cytosol; Female; Humans; Leupeptins; Macromolecular Substances; Molecular Weight; Protein Conformation; Rats; Receptors, Estrogen; Salts; Uterus